Antibiotic am374 and method of production using streptomyces eburosporeus

ABSTRACT

A new antibiotic is produced, designated AM374, by fermenting under controlled conditions Streptomyces eburosporeus n.s. The new antibiotic is active against gram-positive bacteria and thus is useful in inhibiting the growth of such bacteria wherever they may be found. The invention also relates to a novel use of the new antibiotic as a growth promoter for animals.

United States Patent Kunstmann et al.

[ 5] Oct. 24, 1972 ANTIBIOTIC AM374 AND METHOD OF PRODUCTION USING STREPTOMYCES EBUROSPOREUS Inventors: Martin Paul Kunstmann, Pearl River, N.Y.', John Norman Porter,

Ramsey, NJ.

Assignee: American Cyanamid Company,

Stamford, Conn.

Filed: April 6, 1970 Appl. No.: 26,101

Related US. Application Data Continuation-impart of Ser. No. 815,652, April 14, 1969, abandoned.

US. Cl 3....424/118, 424/119 Int. Cl. ..A6lk 21/100 Field of Search ..424/1 18, i 19 [56] References Cited UNITED STATES PATENTS 3,338,786 8/1967 Kunstmann et al ..424/1 18 3,377,243 4/ 1968 De Voe et al ..424/1 18 Primary Examiner-Albert T. Meyers Assistant Examiner-Daren M. Stephens Attorney-Norton S. Johnson [57] ABSTRACT The invention also relates to a novel use of the new antibiotic as a growth promoter for animals.

6 Claims, 1 Drawing Figure ANTIBIOTIC AM374 AND METHOD OF PRODUCTION USING STREPTOMYCES EBUROSPOREUS This application is a continuation-in-part of our 5 (l 96 6 Media usedin the gidy were selected from copending application Ser. No. 815,652, filed Apr. 14, those recommended by Pridham et al., [A Selection 1969, now abandoned. of Media for Maintenance and Taxonomic Study of I This invention relates to a new antibiotic, to its Srreptamyces- Antibiotics Annual (l95 l9 5 7), PP- production by fermentation, to methods for its 947-95315 for the cultivation of streptomycetes. recovery and concentration from crude solutions, to Details are recorded in Tables l-IV, and a general processes for its purification and to the preparation of description of the culture is given below. Underscored its salts. descriptive colors were taken from Jacobson et al.,

The invention also relates to an improved animal [Color Harmony Manual 3rd Ed. (1948)]. feed composition containing the new antibiotic which Amount of Growth w i is effective in accelerating the growth rate of animals Moderate on most media; good on potato dextrose such as chicks, turkey poults, ducklings, etc., as well as agar; light on Czapeks solution agar. young cattle, horses, swine, dogs, and sheep. In this AerialMy and/or en masse Spore Color aspect of the invention an effective amount of the new Aerial mycelium whitish most media; becoming antibiotic is preferably added to a nutritionally Iv ry t0 LL Ivory in areas of sporulationbalanc-ed animal feed and fed to the animals to increase Soluble Pigments the growth rate thereof. Alternatively, the new an- Yellowish on se e i none on Clzapeks solutibiotic may be added to drinking water for ingestion by lion, asparagine-dexlrose and inorganic salts'starch the animals as an aid in controlling infectious diseases. ga

The present invention includes within its scope the Reverse Color antibiotic in dilute forms, as crude concentrates, and in In yellowish Shades on most mediacrystalline forms. These novel products are active MiSCeuaneOuS Physiological Reactions against a variety of microorganisms including grams gt dpged t0 lP' in orsanicnitrate positive bacteria. The effects of the new antibiotic on broth; complete liquefaction of gelatin in 7 day specific microorganisms, together with its chemical and melanin not produced on peptone-iron agar. Carbon physical properties, differentiate it from previously urce u zation c ording to the Pfidham e1 dcscribcd antibiotics. Bact. 56: 107-114, (1948)] method as follows: fair to The new antibiotic, which we have designated good utilization of l-arabinose, (l-fructose, i-inositol, AM374, is formed during the cultivation under conlactose, d-mannitol, d-melibiose, salicin, d-trehalose, dtrolled conditions of a new streptomycete isolatedfrom xylose; poor to non-utilization of l-rhamnose, adonitol, a soil sample collected in Utah. A viable culture of the d-melezitose, d-raffinose and sucrose. new microorganism has been deposited with the Cul- Micromorphology ture Collection Laboratory, Northern Utilization A rial my lium sparse, gi ing ris t l ng tangled Research and Development Division, United States Deand flcxuous chains of elliptical to elongate spores 0.5 partment of Agriculture, Peoria, Illinois and has been to 0.6 p. X 0 t0 I SPore Surface Smooth, as deteradded to its permanent collection. It is freely available ned by electron microscopy. in this repository under its Accession No. NRRL 3582. On the basis of the general characteristics observed,

The description and identification of this new the microorganism becomes a member of the genus microorganism, maintained in the culture collection of Streptomyces. A comparison of the culture NRRL 3582 Lederle Lab rat ri P a lRiv r, N w York,wa5 was made with all available reference specimens of plied by Dr. H. D. Tresner of these laboratories. other streptomycetes having similar basic taxonomic Observations were m eft ew mra r al yshlaa a rfsat rss Tl9l2i9i as, vs a .sbs in TABLE I cal and morphological features of the culture in accordance with the methods detailed by Shirling et al., [Methods for Characterization of Streptomyces Species, lntemat. Joum. of Syst. Bacteriol. 161 3l3340,

Cultural Characteristics of Streptomyces eburosporeus n.s. NRRL 3582 Incubation: 14 days; Temperature: 28 C.

Amount Medium of growth Aerial mycelium and/or spores Soluble pigment Reverse color Remarks Czapeks Solution Light- Aerial mycelium whitish, becoming Ivory (2 db) None Lt. Melon Growth restricted.

Agar. to Lt. Ivory '(2 ea) in sporulation zones. Yellow sporulation light. (3 ea) Asparaginc Dextrose Moderate" Aerial mycelium whitish, sparse. No sporulation do Bamboo Growth restricted; colo y Agar. (2 fb). edge serrate. Hickey and Tresncr's do Aerial mycelium whitish, becoming Ivory (2 db) Yellowish; light... Lt. Amber Surface growth papillose.

Agar. to Lt. Ivory (2 ea) in sporulation zones. Sporu- (3 ic).

lation light. Yeast Extract Agar do Aerial mycelium whitish; trace of sporulation do do Surface rugose. Kustors Oatllake do Aerial mycelium whitish, becoming Ivory (2 db) Yellowish-brown; Lt Melon Moderate starch hydrolysis.

Agar. to Lt. Ivory (2 ca) in sporulation Zones. Sporulight. Yellow lotion light. (3 ea). Tomato Paste Oatdo Aerial mycelium whitish, becoming Ivory (2 db) Orange-yellow; Lt. Amber Surface papillose.

meal Agar. llbotLt. lvolrty (2 ea) in sporulation zones. Sporumoderate. (3 lo) a ton 1g Potato Dextrose Good. Aerial mycelium whitish, sparse. Becoming do do Starch hydrolysis llght.

Agar. Ivory (2 db) in Lt. Ivory (2 ea) in sporulation Colony surlace rugose to zones. sporulation light. rimose. I Bennett's Agar Moderate" Aerial mycelium whitish, sparse. Trace of Yellowish; light do Surface wrinkled and sporulation. papillose. Inorganic Salts do Aerial mycelium whitish, becoming Ivory (2 db) None Lt. Melon Starch Agar. to Lt. Ivory (2 ea) in sporulation zones. Sporu- Yellow lation light. (3 ea).

TABLE II Micromorphology of Streptomyces eburosporeus n.s. NRRL 3582 Medium Aerial mycelium and/or sporiierous structures Spore shape Spore size Spore surface Kusters Oatfiake Aerial mycelium sparse, giving rise to long tangled Elliptical to Spores0.50.6px Smooth as determined by electron Agar. and fiexuous chains of spores. ongate. 1.0-1.3,4. microscopycommon with NRRL 3582; however, when the litera- Sucrose ture descriptions of these related species were ex- 2???? amined, certain basic differences were found. These Dexzmse 3 are set forth in Table v. In addition to these dif- Negative control 0 ferences, NRRL 3582 generally produces a markedly more restricted growth on most media and its sporulation tends to be more sparse. *3-Good Utilization It will be noted in Table V that the sporophore l 9" morphology of NRRL 3582 is of the Reczus-Flexibilis g gg fj g' 'gg type of Pridham et al., [Appl. Microbiol. 6: 52-79, (1958)]Spira. This is a very fundamental difference, and it alone is TABLEV adequate to distinguish between the organisms. When considered together with the other differences in- Companson ffstreptomyces NRRI: 3582W1th dicated, NRRL 3582 stands out sufficiently from the bldoflavusand odorfer others to be regarded as a separate species. smpwmyces In keeping with the ivory color of the spores NRRL albidvflflvS f" produced by the organism, the name Streplomyces eburosporeus n.s. is proposed as an appropriate descriptive Aerial ith t mycelium Sporophores Sporophores Sporophores p and/or long, short. long, straight,

Sporeflexuous and spiral-forming. branching; Bearing tangled. forming spirals Branches TABLE Spore Elliptical Spherical Spherical shape to elongate Miscellaneous Physiological Reaction of Streptomyces Gem complew Rapid slow eburosporeus lLS. NRRL 3582 liqueliquegaction liquefaction liquefaction faction Temperature; 28C Soluble Mostly Mostly Mostly pigments yellowish yellowish brownish Medium Incubation Amount of Physiological Reaction Gmwlh Gmwlh YCHO- GTOWlh Growl! cream Period Growth on Aspawish; aerial Aerial mycelium colored to broraginemycelium becoming wnish. Aerial Dextrose whitish whitish-yellow. mycelium abun- Organic 7 days Good No nitrate reduction Agar Sparse d ream- Nitrate colored. Broth Organic 14 days Good No nitrate reduction. Nitrate Broth Gem, 7 days Good complete liquefacnm It 18 to be understood that for the production of the p 24 hours Moderate Melanin Produced new antibiotic the present invention is not limited to 2; this particular organism or to organisms fully answering the above growth and microscopic characteristics which are given for illustrative purposes. in fact, it is desired and intended to include the use of mutants TABLE IV produced from the described organism by various means, such as x-radiation, ultraviolet radiation, Carbon Source Utilization Pattern of Streptomyces nitrogen mustard, phage exposure and the like.

eburosporeus n.s. NRRL 3582 THE FERMENTATION PROCESS Incubation: 10 days Temperature: 28C.

Carbon Source Utilization m-CwcwwwtowO Cultivation of the organisms S. eburosporeus may be carried out in a wide variety of liquid culture media. Media which are useful for the production of the novel antibiotic include an assimilable source of carbon such as starch, sugar, molasses, glycerol, etc.; an assimilable source of nitrogen such as protein, protein hydrolysate, polypeptides. amino acids, corn steep liquor, etc.; and inorganic anions and cations, such as potassium, sodium, calcium, sulfate. phosphate, chloride, etc. Trace elements such as boron, molybdenum, copper, etc.; are supplied as impurities of other constituents of the media. Aeration in tanks and bottles is provided by forcing sterile air through or onto the surface of the fermenting medium. Further agitation in tanks is provided by a mechanical impeller. An antifoaming agent, such as 1 percent octadecanol, in lard oil may be added as needed.

INOCULUM PREPARATION Shaker flask inoculum of S. eburosporeus is prepared by inoculating 100 milliliters of sterile liquid medium in 500 milliliter flasks with scrapings or washings of spores from an agar slant of the culture. The following medium is ordinarily used.

Molasses 20 grams Glucose grams Bactopeptone 5 grams Water to 1,000 milliliters.

The flasks are incubated at a temperature from 25-29 C., preferably 28C., and agitated vigorously on a rotary shaker for 30 to 48 hours. These 100 milliliter inocula are used to inoculate 1 liter and 12 liter batches of the same medium in 2 liter and liter glass fermentors. The inoculum mash is aerated with sterile air while growth is continued for 30 to 48 hours. These batches of inocula are used to inoculate tank fermentors.

TANK FERMENTATION For the production of the antibiotic in tank fermentors the following fermentation medium is preferably used.

Soy bean flour 10 grams Cerelose 10 grams Sodium chloride 5 grams Calcium carbonate 1 gram Distillers solubles 5 grams from corn Water to 1,000 milliliters.

Each tank is inoculated with 3 to 10 percent of inoculum made as described above. Aeration is supplied at the rate of 0.5-1 .0 liter of sterile air per liter of broth per minute and the fermenting mixture is agitated by an impeller driven at 200-400 r.p.m. The temperature is maintained at -29C., usually at 28C. The fermentation is ordinarily continued for 65-90 hours, at which time the mash is harvested.

PURIFICATION PROCEDURE charcoal may be eluted by stirring the charcoal for I about one-half hour with 40 percent aqueous acetone adjusted to pH 2 with concentrated sulfuric acid, using a volume of eluate equal to about one-quarter of the original beer volume. The eluate is concentrated under reduced pressure to an aqueous phase equal to about One-twentieth o t w tekw qms hr.i 1 hi phase is adjusted to about 5.5 with barium hydroxide and the barium sulfate precipitate which forms is removed by filtration. The adjusted solution (filtrate) is further concentrated to about 400-800 ml. This concentrate is then slurried with acidified alumina (using about one-fifth of the amount of alumina in grams as compared to the volume of concentrate) and this slurry is poured onto a suitable column of acidified alumina (using approximately ten times the amount of alumina used in the charge) wet-packed in methanol. The antibiotic activity is eluted from the column with aqueous methanol (usually 25-50 percent) and appropriate active fractions are collected. The active fractions are combined and concentrated to a small volume (one liter or less) under reduced pressure. The pH of this concentrate is adjusted to about 6.0 to 6.5 with barium hydroxide. Again, the barium sulfate precipitate which forms is removed by filtration and the clear filtrate lyophylized to yield the (crude) antibiotic AM374. This lyophylized material is now further purified by column chromatography on a suitable ion exchange resin, as for example, CM Sephadex C-25 (I-I form). The column may be eluted using dilute sulfuric acid solutions. The eluate containing the antibiotic activity, located by absorption at 280 mu, is adjusted to about pH 6.3 with barium hydroxide. The barium sulfate precipitate which forms is removed by filtration and the filtrate is concentrated to a volume of about 30-80 milliliters. The 'pH of this solution is adjusted to about 8.0 with IR45 (OI-1') resin. The resin is removed by filtration and the filtrate is added to a larger quantity of acetone with stirring and the precipitate which forms is recovered by filtration. The precipitate is washed with acetone and dried at moderate temperature under reduced pressure to yield antibiotic AM374 in the base form.

PHYSICAL CHARACTERISTICS Carbon 54.54 Hydrogen 6.10 Oxygen 29.01 Nitrogen 7.60 Chlorine 2.47

The material does not have a definitive melting point, decomposing slowly above 250C. Optical rotation is ==102(i2.9) (C=1.045 in water). Ultraviolet maxima occur at:

282 m;:.. (E 5, 42) in acidic solutions 282 mp. (51%, 42.5) in neutral solutions 305 mu. (E jig 51.5) in basic solutions sh260 m;:.. (E {3 92) in basic solutions An infrared absorption spectrum of AM374 base in a KBr pellet, prepared in a standard manner, exhibits characteristic absorption at the following wavelengths expressed in microns: 3.0, 3.43, 6.0, 6.20, 6.30, 6.67, 6.88, 7.05, 7.22, 7.35sh, 7.52, 7.67, 8.2, 8.65sh, 8.85, 9.45, 9.75, 9.90, 10.45sh, 11.07, 11.5, 12.0, 12.35, 2.; 15+...

v The infrared curve is showii in FIG. 1 of the accoitipanying drawing.

AM374 antibiotic shows the following R, values in the solvent systems indicated below using Bacillus subtilis pH 6.0 or Corynebacterium xerosis as detection organisms:

R, Value Solvent System 0.90 5% aqueous NH,CI 0,20 pyridine 2 parts u=collid1ne 2 putts sec-butnnol 1 part water 1 part TABLE V] In Vitro Antimicrobial Activity of Antibiotic AM374 and Its Hydrolysis Product* Minimal lnhibitory Concentrations (Micrograms per ml.)

Antibiotic Hydrolysis Am374 Product Staphylococcus aureus 3.1 6.2 ATCC 65381 Staphylococcus aureus 3.1 3.1 No. 69 Staphylococcus aureus, 6.2 6.2 Rose ATCC 14154 Staphylococcus aureus, 6.2 6.2 Smith ATCC 13709 Streptococcus pyogenes 0.62 1.25 C203 Streptococcus faecalis 3.1 6.2 ATCC 8043 Streptococcus sp., 6.2 6.2

nonhemolytic No. 11 Streptococcus sp., 6.2 6.2

- B-hemolytic No. 80

M ycobacterium smegmatis 250 125 ATCC 607 Salmonella typhosa 250 250 ATCC 6539 Proteus vulgoris 250 250 ATCC 9484 Escherichia coli 250 250 U31 1 Escherichia coli 250 250 Klebsiella pneumoniae 250 250 Strain AD Enterobacler aerogenes 250 250 N0. 75 Pseudomanas aeruginosu 250 250 ATCC 10145 Using agar dilution method.

such microorganisms. The new antibiotic can be expected to be usefully employed for treating or controlling such infections by topical application or parenteral administration.

The usefulness of the new antibiotic is demonstrated by its ability to control systemic lethal infections in mice. The new antibiotic shows high in vivo antibacterial activity in mice against Staphylococcus aureus, strain Smith; Staphylococcus aureus, strain Rose; Streptococcus pyogenes, C203: and Diplococcus pneumoniae, SVl when administered by a single subcutaneous dose to groups of Carworth Farms CF-l female mice, weight about 20 grams, infected intraperitoneally with a lethal dose of these bacteria in 10', 10, 10" and 10 trypticase soy broth TSP dilutions, respectively, of a five hour TSP blood culture.

Table VII, below, illustrates the in vivo antibacterial activity of AM374 while Table VIII, following, illustrates the in vivo antibacterial activity of the hydrolysis product.

TABLE VII Staphylacoccus oureus strain Rose Staphylococcus aurcus strain Smith Streptococcus pyagenes C203 Y Dosage mgJkg. body wt.

Diplococcus pneumonia:

SV I

I Of the infected non-treated control mice 95-10096 died within 5 days after infection.

TABLE VIII 2 Alive/total mice, 14 days after infection Dosage mgJkg. body wt.

Staphylococcus au reus strain Smith As indicated earlier, the novel antibiotic is also highly effective in promoting the growth rate of animals. In carrying out this aspect of the present invention, an effective amount of the novel antibiotic is added to drinking water or preferably a nutritionally balanced animal feed and is fed to the animals to increase the growth rate thereof.

The invention will be described in greater detail in conjunction with the following specific examples.

EXAMPLE 1 lnoculum Preparation A typical medium used to grow the primary inoculum was prepared according to the following formula:

Molasses 20 grams Glucose 10 grams Bactopcptone grams Water to 1,000 milliliters I The washed or scraped spores from an agar slant of S. eburosporeus were used to inoculate two flasks containing 100 milliliters each of the above medium in 500 milliliter flasks. The flasks were placed on a rotary shaker and agitated vigorously for 48 hours, at 28C. The resulting flask inoculum was transferred to a 5 gallon glass'fermentor containing 12 liters of sterile medium. The glass fermentor was aerated with sterile air while growth was carried out for about 48 hours, after which the contents were used to seed a 300 liter tank fermentor.

EXAMPLE 2 Fermentation A fermentation medium was prepared according to the following formula:

Soy bean flour grams Cerelose 10 grams Sodium chloride 5 grams Calcium carbonate l gram Distillers soluble 5 grams from corn Water to 1,000 milliliters.

The fermentation medium was sterilized at 120C. with steam at pounds pressure for 45-60 minutes. The pH of the medium after sterilization was 6.6. Three hundred liters of sterile medium in a 400 liter tank fermentor was inoculated with 12 liters of inoculum such as described in Example I, and the fermentation was carried out at 28C. using Hodag LG-8 oil as a'defoaming agent. Aeration was supplied at the rate of 0.5 liter of sterile air per liter of mash per minute. The mash was agitated by an impeller driven at 300revolutions per minute. At the end of approximately 70 hours of fer mentation time the mash was harvested.

EXAMPLE 3 Isolation and Purification 300 Liters of fermented mash were filtered with about 2 percent (w/v) of diatomaceous earth filter aid and the filter pad was washed with about 30 liters of water. The fermented mash was previously adjusted to pH 6.0 with sodium hydroxide. The antibiotic activity in the pooled filtrate and water wash was adsorbed onto 900 grams of Darco G60 and colored impurities were removed from the charcoal suspension by stirring with about 30 liters of 40 percent aqueous acetone. The activity was eluted from the charcoal by stirring with 75 liters of 40 percent aqueous acetone adjusted to pH 2.0 with concentrated sulfuric acid. The suspension was filtered, the eluate was concentrated under reduced pressure to about 4 liters of aqueous phase and the pH was adjusted to 5.2 with barium hydroxide. The bariumsulfate precipitate was removed by filtration and the filtrate further concentrated to about 700 ml. This concentrate was slurried with 100 g. of acid-treated alumina and poured onto a column consisting of 1 kg. acid-treated alumina in methanol. The acid-treated alumina was prepared by acidifying an aqueous slurry of Merck alumina with concentrated sulfuric acid until-a constant pH of 3.0 was obtained, filtering, washing with water and methanol, and air drying. The column was then eluted with 5 liters of methanol followed by 60 liters of 50 percent aqueous'methanol followed by 10 liters of 25 percent aqueous methanol. Appropriate fractions containing antibiotic AM374, determined by bioassay against C. xerosis, were combined and concentr'ated under reduced pressure to about 500 m1. and the pH was adjusted to 6.2 with barium hydroxide. The barium-sulfate precipitate was removed by filtration and the filtrate lyophilized to give 5.1 grams of crude AM374 (about 30 to 50 percent pure).

The 5.1 grams of crude AM374 thus obtained was dissolved in 30 ml. of water and applied to a column of CM Sephadex C-25 (H form). The Sephadex (250 g.)

was prepared for use by being slurried in about 3 liters of water and the pH adjusted to 2.0 with concentrated H The excess water was decanted and the resin washed several times with water. The slurry was poured into a column (7.6 cm. LD.) and washed with an additional 5 liters of water removing the excess acid.

The column was eluted with-a gradient between 4 liters of water and 4 liters of water adjusted to pH 1.4 with concentrated sulfuric acid, followed by an additional 4 liters of water adjusted to pH 1.4. Fractions (about ml. each) were collected and bioactivity (C.

xerosis) and ultraviolet absorbance (280 mp.) were measured on 1 to 100 dilutions of these fractions.

Fractions 58 through 80 were shown to contain the majority of the desired antibiotic and were combined into one pool. The pH of this pool was adjusted to 6.3 with barium hydroxide and the precipitated barium sulfate was removed by filtration with the aid of diatomaceous earth. The filtrate was concentrated to about 200 ml. and lyophilized yielding about 1.5 grams of crude antibiotic. The crude antibiotic was dissolved in 50 ml. of water and the pH of the solution was adjusted to about 8.2 with IR45 (OH form). The suspension was filtered and the filtrate was lyophilized. The lyophylizate was dissolved in 40 ml. of water'and the antibiotic was precipitated upon the addition of 400 ml.

of acetone. The crystalline precipitate was collected by ethanolic acetone and drying the precipitate under high vacuum (10' mm.) at 100C.for two days. The chemical analysis of this product and the other physical and biological properties of the new antibiotic have already been described.

EXAMPLE 4 Conversion of AM374 Base into AM374 Sulfate One hundred and fifty milligrams of AM374 base was dissolved in 60 ml. of warm methanol to which was added one drop of a 1:1 methanol-concentrated sulfuric acid solution. The resulting precipitate was filtered and washed with some methanol and acetone to give 98 mg. ofAM374 sulfate.

Alternatively, AM374 sulfate was prepared by adjustin g an aqueous solution of AM374 base to about pH 6.3 with sulfuric acid, followed by precipitation with acetone.

A microanalytical sample was prepared by precipitation from aqueous acetone and drying the precipitate under high vacuum (10'' mm.) at 100C. for 2 days. AM374 sulfate prepared in this manner contains the elements carbon, hydrogen, oxygen, nitrogen, sulfur and chlorine in substantially the following percentages by weight:

Carbon 51.68 Hydrogen 5.85 Oxygen 30.25 Nitrogen 7.02 Sulfur 1.36 Chlorine 2.27

Optical rotation is [01],, 104 2.8) (C=1.075 in water). Ultraviolet maxima occur at:

282 mp. (E 1 44) in acidic solutions 282 mu. (E 1%,, 43) in neutral solutions 305 mu. (E 1%,, 48) in basic solutions sh260 mu. (E 12? 85) in basic solutions An infrared absorption spectrum of AM374 sulfate in a KBr pellet, prepared in a standard manner, exhibits characteristic absorption at the following wavelengths expressed in microns:

3.0, 3.45, 5.75sh, 5.90sh, 5.99, 6.03, 6.20, 6.32, 6.67,

6.78sh, 6.87, 7.05, 7.20, 7.53, 7.65, 8.25, 8.65sh, 8.87,

9.45, 9.73, 9.88sh, 10.45sh, 11.05, 12.0, 12.35, 13.3, 14.4.

EXAMPLE 5 Conversion of AM374 Base into AM374 Chloride Two hundred milligrams of AM374 base was dissolved in ml. of 0.1N hydrochloric acid and concentrated to about 2 m1. under reduced pressure at'which time a partially microcrystalline precipitate formed. This was filtered, the precipitate redissolved in a minimum of water and reprecipitated with acetone. This was filtered and washed with some additional acetone to give 1 16 mg. of AM374 chloride.

A microanalytical sample was prepared by precipitation from aqueous acetone and drying the precipitate under high vacuum (10 mm.) at 100C. for 2 days. AM374 chloride prepared in this manner contains the elements carbon, hydrogen, oxygen, nitrogen and chlorine in substantially the following percentages by weight:

Carbon 5210 Hydrogen 6.22 Oxygen 28.03 Nitrogen 7.26 Chlorine 634 Optical rotation is [11],, =106 (i2.8) (C=1.057 in water). Ultraviolet maxima occur at:

282 m;.:.. (E 1 31,, 42) in acidic solutions 282 m;;.. (E V1,, 39.5) in neutral solutions 305 mu. (E 1%,, 52) in basic solutions sh260 nm. (E 129,, 92) in basic solutions An infrared absorption spectrum of AM374 chloride in a KBr pellet, prepared in a standard manner, exhibits characteristic absorption at the following wavelengths expressed in microns: 3.0, 3.27, 3.40, 5.75sh, 5.90sh, 5.98, 6.15, 6.28, 6.65,

, EXAMPLE 6 Acid Hydrolysis Product of AM374 Base One gram of AM374 antibiotic base was dissolved in 7.5 ml. of boiling water. To this was added 1.25 ml. of 5N hydrochloric acid and the resulting solution boiled an additional 2 to 3 minutes. An additional 2 ml. of 5N hydrochloric acid was added and the solution was cooled. This was filtered and the precipitate washed with a total of 3 ml. of 5N hydrochloric'acid. It was redissolved in water and concentrated to aresidue.

This was repeated twice more after which the residue was precipitated from aqueous acetone to give 516 mg. of acid hydrolysis product.

A rnicroanalytical sample was prepared by precipitation from aqueous acetone and drying the precipitate under high vacuum (10' mm.) at C. for 2 days. The AM374 hydrolysis product prepared in this manner contains the elements carbon, hydrogen, oxygen, nitrogen and chlorine in substantially the followmg percentages by weight:

Carbon 52.54 Hydrogen 5.81 Oxygen 25.52 Nitrogen 8.32 Chlorine 7.1 1

Optical rotation is [01],, =69 (136) (C=0.839 in water). Ultraviolet maxima occur at:

280 mp..'(E 1?... =46) in acidic solutions 280 mu. (E Zm. 51.5) in neutral solutions EXAMPLE 7 The basal diet employed in the following poultry tests had the following composition.

Poultry Basal Diet Ingredient Gms./Kg.

Ground yellow corn 514 Soybean oil meal (44%) 300 Corn gluten meal 50 Menhaden fish meal (60%) 50 Fat 40 Dehydrated-alfalfa meal (17%) 20 Ground limestone 5 Dicalcium phosphate 12 Sodium chloride 3 .3 l4 Trace minerals V V 1 I H d. has ultraviolet maxima at: 282 mp. E {1, 42) 5 3 in acidic solutions, 282 mu. (E Ri 42.5) in neutral solutions, 305 m,u.. (E ij 51.5) in basic solutions, sh260 mu. (E 13 92) in basic solu- Trace minerals are manganese (6.0%), iodine (.l2%), iron (2.0%), 5 tjons;

copper (.2%),zinc (2.0%),cobalt (.O2%),and calcium (25.5%). 6 o Vitamin pre-mix, per kg. of feed, contains l25 mg. butylated hydroxy has an opPlcal rotatlon' [(11.11 102 161116116. 500 mg. DL-methionine, 3300 w. Vitamin A, 1100 1.u. (C=l.0451n water);and Vitamin D 2.2 LU. Vitamin E, ll mcg. Vitamin B 4.4 mg.

riboflavin, 27.5 mg. niacin, 8.8 mg. pantothenic acid, 500 i'hg. choline has a chafractensnc Infrared absorptlon Spectrum chloride, 1.43 mg. r6116 acid, and 1.1 mg. menadione sodium bisulfite as shown in FIG. 1.

2. A compound as defined in claim 1, antibiotic AM374, in its essentially pure form. Day-old ch1cks (6 males and 6 females per group) purchased from a commelrcial Supplier are q f in antibiotic AM374, according to claim 1, and salts heated brooders and held in a chick room maintained thereof at about 24C. All chick groups are weighed at the start Acid hydrolysis product of antibiotic AM374 as of the tests and on termination at days. Feedand definedin claim lwhich acid hydrolysis product water are supplied ad llbitum. The basal diet described has the following elemental analysis. C, 5 4; H

above is used for all tests. Treatments used were (a) un- 5 O 25.52. N c1 treated controls (b) 10 PPM AM374 (c) 2 b. has ultraviolet maxima at: 28O mu. 0E :2 46) AM374. Data obtained are reported below where it can 20 in acidic Solutions 280 my, CE is :5 5) in be seen that at both treatment levels AM374 showed a neutral Solutions mph i 1% 86) in'basic marked improvement in the growth of treated birds. solutions, Sh260 my CE 2,51 5133) in basic Solu Growth and Feed Efficiency by Chicks ions;

c. has an optical rotation: [11],, 69 (i3.6) Receiving Rations Containing AM374H (C=0 839 i wat d v Surd. exhibits characteristic infrared absorption at the Feed Level E py e viva} lmpfovmem 'following wavelengths expressed in microns: 3.0, 3.30511 3.43 5.75811 5.92511 6.0 6.15sh 6.25 2: 1 I) A :22 1.23 53:38 6.48sh, 6.68, 6.85, 7.03, 7.18, 7.5, 7.67, 7.96sh,

8 30 8.55sh 8.86 9.15 9.45 9.9 10.45sh 11.1 N B 288 L 5 3 o 9 Q 9 a a a a Ala/$7411 2 302 1.63 13520 4.9 7.4 11- 5. A process which comprises cultivating Streptomyces eburosporeus NRRL 3582 in an aqueous nutrient We claim; medium under aerobic conditions until substantial an- 1. Antibiotic AM374, a compound hi tibacterial activity is imparted to said medium by the a. is effectiv i i hibiti h growth f gram posi production of a compound as character zed in claim 1.

tive bacteria, and in its essentially pure crystalline A Process whch cmpnse s cumvatmg P form; myces eburosporeus NRRL 3582 in an aqueous nutrient b i dil l bl i water and dimethyl sulfoxide medium under aerobic conditions until substantial anand moderately soluble in methanol and ethanol 40 tibacterial activity is imparted to Said medium y h and relatively insoluble in other common organic Production of a compound as characterized in Claim 1 l t and isolating the compound therefrom.

c. has the following elemental analysis: C, 54.54; H,

6.10; O, 29.01;N, 7.60 ;'Cl, 2.47;

3. A compound selected from the group consisting of UNITED STATES PATENT CFFICE CERTIFICATE OF CORRECTION Patent NO. j,700.Z68 Date October 24. 1972 Inventor(s) Martin Paul 'Kunstmann and John Norman Porter It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below: I

Column 5, line 18, before "S ira insert whereas that of the other two species is Column 4, Table V, under the heading Streptomyces NRRL", the third entry, "Complete liquegaction" should read Complete liquefaction Column 7, Table VI, the last entry in the first column,

"Pseudomanas" should read Pseudomonas Column 14, Claim line 19, "cEi'Z +6) should read 46) line 20 "CEifi 51.5 should read ='5l.5) -5 line 22, "CE-51in 1 45)" should read (Eii 145) line 29, "7.96sh". should read 7.95sh

Signed and sealed this 27th day of March 1973.

(SEAL) Attest:

EDWARD M.FLETCHER,JR. ROBERT GOTTSCHALK Attesting Officer Commissioner of Patents FORM powso Ho's-9) USCOMM-DC 60376-F'09 .5. GOVIINIIEIIT "INYIIG OFFICE 7 l9" O-'3fi-33 

2. A compound as defined in claim 1, antibiotic AM374, in its essentially pure form.
 3. A compound selected from the group consisting of antibiotic AM374, according to claim 1, and salts thereof.
 4. Acid-hydrolysis product of antibiotic AM374 as defined in claim 1, which acid-hydrolysis product a. has the following elemental analysis: C, 52.54; H, 5.81; O, 25.52; N, 8.32; Cl, 7.11; b. has ultraviolet maxima at: 280 m Mu . cE1cm.1 46) in acidic solutions, 280 m Mu . cE1cm.1 51.5) in neutral solutions, 300 m Mu . (Ecm.1 86) in basic solutions, sh260 m Mu . cE1cm.1 143) in basic solutions; c. has an optical rotation: ( Alpha )D25 -69* ( + or - 3.6*) (C 0.839 in water); and d. exhibits characteristic infrared absorption at the following wavelengths expressed in microns: 3.0, 3.30sh, 3.43, 5.75sh, 5.92sh, 6.0, 6.15sh, 6.25, 6.48sh, 6.68, 6.85, 7.03, 7.18, 7.5, 7.67, 7.96sh, 8.30, 8.55sh, 8.86, 9.15, 9.45, 9.9, 10.45sh, 11.1, 11.55sh, 11.9, 13.3, 14.4.
 5. A process which comprises cultivating Streptomyces eburosporeus NRRL 3582 in an aqueous nutrient medium under aerobic conditions until substantial antibacterial activity is imparted to said medium by the production of a compound as characterized in claim
 1. 6. A process which comprises cultivating Streptomyces eburosporeus NRRL 3582 in an aqueous nutrient medium under aerobic conditions until substantial antibacterial activity is imparted to said medium by the production of a compound as characterized in claim 1 and isolating the compound therefrom. 